PROJECT SUMMARY/ABSTRACT A major translational hurdle for hematopoietic stem cell (HSC) biology is the in vitro generation of bona fide, transplantable hematopoietic stem cells. As adult bone marrow HSCs are relatively quiescent, and not physiologically required to expand in large numbers, attempts at in vitro expansion have resulted in limited self- renewal, inability to provide multi-lineage engraftment, and cell death. An alternative approach has been to reprogram cells with early embryonic attributes to HSCs, but many times the cells generated are too immature and unable to provide long term engraftment. To improve current attempts of generating HSCs in vitro, it is imperative we understand how HSCs are initially formed in the embryo. It is now well accepted that the precursor to an HSC is a specialized hemogenic endothelial cell, that undergoes an endothelial to hematopoietic fate transition. While the first HSCs arise from an endothelial-to-hematopoietic (EHT) transition within a subset of endothelial cells, during a narrow window of development, it is still unclear why certain endothelial cells undergo the transition and not others, despite similar gene expression patterns and anatomic location; and it is also unclear why the process only occurs during a narrow developmental time window. While the factors regulating EHT are beginning to be elucidated, the production of HSCs solely from endothelium has yet to be achieved, suggesting there may be other cell requirements yet to be discovered. One major well studied hemogenic vascular site is the dorsal aorta, where in addition to hemogenic endothelial populations, there exists an extensive mesenchymal cell population underlying the hemogenic vasculature. Due to the previous lack of restricted markers for the mesenchymal layer, studying the contribution of this compartment to hemogenic endothelium and HSC emergence has not been previously possible. Recently we have identified a mural cell marker that is specifically expressed in mesenchymal layer during the hemogenic window. Capitalizing on this new insight, we plan to fully examine the role of the sub-aortic mesenchyme in the development of HSCs from the endothelium. We hypothesize that the underlying mesenchyme plays a regulatory role in controlling hemogenic endothelial cell number and conversion to hematopoietic fate. This work will shed light on the critical steps in HSC emergence and specification, laying the groundwork for large- scale in vitro generation of HSCs.